As a method of preparing retinal pigment epithelial cells from human pluripotent stem cells, (i) a method in which cells are left to spontaneously develop into retinal pigment epithelial cells by extracting bFGF from a medium component for culturing human ES cells (spontaneous differentiation method), (ii) a method based on coculture with mouse mesenchymal cell line PA6 (SDIA method), (iii) a method in which SFEB method including culturing human ES cells as a floating aggregate in a serum-free medium is combined with Nodal signal inhibitor Dkk1 and Wnt signal inhibitor LeftyA (SFEB/DL method) and the like are known. However, (i) spontaneous differentiation method (U.S. Pat. No. 7,736,896 etc.) is associated with a problem of marked variation in the differentiation induction efficiency depending on the line of human ES cell and the like to be a starting material. (ii) SDIA method (Proc Natl Acad Sci USA. 2002 Feb. 5 99(3) etc.) is feared to cause a safety problem due to virus contamination etc. resulting from the use of a cell of a mouse, which is an xenogeneic animal, even though the differentiation induction efficiency does not rely much on the difference in the line of human ES cell and the like to be used, and constant differentiation induction into retinal pigment epithelial cell is possible. (iii) SFEB/DL method (PNAS Aug. 9, 2005 vol. 102 no. 32 11331-11336, Nat Biotechnol. 2008 Feb. 26(2)215-24 etc.) reported thereafter is more suitable as a method for producing cells for cell transplantation therapy, as compared to the aforementioned (i) and (ii), since it shows comparatively small variation in the differentiation induction efficiency among human ES cell lines, and does not require separate preparation of xenogeneic cells.
During the process of examining clinical application of the SFEB/DL method, the present inventors have found that differentiation inducing factors including recombinant protein Dkk1 corresponding to the Nodal signal inhibitor, and recombinant protein LeftyA corresponding to the Wnt signal inhibitor can be substituted by low-molecular-weight compounds CKI-7 and SB-431542, respectively (SFEB/CS method; WO2008/087917), and established a method of stably and economically ensuring the supply of a differentiation inducing factor. However, the SFEB/CS method requires a long term of about 40 days before emergence of pigment cells from pluripotent stem cells (Journal of Cell Science 2009 Sep. 1 122(Pt 17) 3169-79), thus posing a major problem in the quality management and economical aspect. In addition, a floating culture has a problem in that the cells are easily lost during medium exchange, thus causing a decrease in the yield of the final product. Furthermore, for transplantation use, a method capable of stably obtaining a population of highly pure retinal pigment epithelial cells by a simple method has been desired